Just how heterocyclic amine (HA) DNA adducts influence mutagenesis is not known with certainty, however, this question is of interest in light of the possible role of DNA adducts and mutations in the carcinogenic process. Previously, we compared the mutation spectra of 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo [4,5- f]quinoline (IQ)in the supF shuttle vector system using repair deficient and proficient human fibroblasts. The predominant mutation induced was a G to T transversion mutation. However, there were differences between the two HAs in their mutation spectra, and the mutation spectra were influenced by DNA repair. We further examined whether there was a correlation between mutation hotspots and adduct hotspots. A polymerase arrest assay thermal cycle sequencing was developed to measure the levels of IQ and PhIP adducts in defined gene sequence. This method was applied to study the levels of IQ and PhIP adducts in the supF gene. IQ and PhIP adducts were found to form only at the guanine base in the supF gene, and the distribution of IQ and PhIP adducts was similar. Notably, adduct hotspots did not correlate with mutation hotpots. These findings emphasize that adduct hotspots are not necessarily mutation hotspots and further that relatively low adduct levels are needed to induce mutations at mutation hotspots.